Lactococcus strains are used as starter cultures for the production of cheeses and fermented milks. Bacteriophage infection of the starter culture remains a serious problem for the cheese industry and can result in a slow or dead cheese vat. Several mechanisms of phage defense have been identified in lactococci. These include adsorption blocking, abortive infection, and R-M systems (26). A report has shown the beneficial effect of using different phage resistance mechanisms in rotation (42). By cloning phage resistance mechanisms from lactococci it would be possible to construct a "cassette" like system consisting of different phage resistance mechanisms.
Restriction-modification (R-M) systems have been found in a wide range of bacteria. At least three different types of R-M systems, type I, II and III, have been found and characterized with respect to their requirement of Mg.sup.2+, ATP and S-adenosyl-methionine (2). The type II R-M system is by far the most simple and best understood of the R-M systems, containing a separate methylase (MTase) which uses S-adenosyl-methionine as the methyl donor and an endonuclease (ENase), both recognizing the same sequence. Today more than 200 different type II R-M systems have been identified and more than 100 of them have been cloned, mainly because of their importance as tools in molecular biology and the important knowledge which is achieved of protein-DNA interactions. The genetic characterization of type II R-M systems shows that the genes for the ENase and the MTase are closely located, although not always in the same orientation. The MTase and the ENase from the same type II R-M system normally do not show any homology to each other at the amino acid level despite the fact that they recognize the same DNA-sequence. Comparisons between type II MTases have shown that strong similarities exist within the group consisting of 5-methylcytosine MTases (m.sup.5 C MTases) and within the group consisting of N4-methylcytosine (m.sup.4 C) and N6-methyladenosine (m.sup.6 A) MTases (29). The m.sup.5 C MTases have about ten common amino acid sequence motifs, whereas the m.sup.4 C and m.sup.6 A MTases share two major common amino acid sequence motifs. In contrast, the ENases have generally very little homology in common, and no strong sequence motifs have been found.
Several plasmids encoding R-M systems have been found in lactococci. However they have not been characterized at the molecular level, but only in vivo by their efficiency in restricting phages (38). Fitzgerald et al. (11) examined eight different strains and found only one type II endonuclease activity, R, ScrFI, in one strain, L. lactis UC503 (originally designated Streptococcus cremoris F). This first type II R-M system, ScrFI, isolated and characterized from a L. lactis strain, has been found to be chromosomally encoded (11, 8). Later Daly and Fitzgerald (6) examined seven strains more from different starter cultures and found that six of the strains had ENase activity similar to R, ScrFI. No other type II ENase activities were found. The ENase from ScrFI recognized 5'-CC.dwnarw.NGG-3'. Two ScrFI MTase-encoding genes have been cloned and characterized, but neither of the two isolated M,ScrFI-carrying clones exhibited any ScrFI ENase activity (8). The nucleotide sequences of the two MTase-encoding genes have been determined (8, 45). Both contained all ten of the predictive motifs normally found in m.sup.5 C MTases. Mayo et al. (32) have reported type II activity, LlaI recognizing the sequence CC.dwnarw.WGG from L. lactis NCDO497, but they did not determine whether it was chromosomally or plasmid encoded.
A type IIS system, LlaI, has been identified on the lactococcal plasmid pTR2030, which also codes for an arbortive infection mechanism (17). The nucleotide sequence of a type IIS MTase, M,LlaI, from the plasmid pTR2030 has been identified and determined (18).
TK5 is a Danish starter culture, which has been used for the production of Cheddar cheese since 1982 (33). This starter culture has a marked resistance to phages as the dairy during 11 years of continuous production did not observe any delay in acidification due to a phage infection, even though phages were isolated from the whey. The starter originates from an old traditional dairy starter culture, which consists of an unknown number of L. lactis strains. Sixty-two bacterial isolates were purified from the TK5 starter. 33 of the isolates were arranged according to their plasmid profiles into six groups of identical or nearly identical profiles; 27 of the isolates showed unique plasmid profiles (22). All isolates have between 5 and 10 plasmids.
In order to identify plasmid-encoded phage resistance in Lactococcus a cotransformation procedure was used. Total plasmid DNA from L. lactis strain W56 isolated from the TK5 starter culture (33) was transformed together with the vector pVS2 (53) into Lactococcus lactis subsp. cremoris MG1614. We selected for the Cm.sup.R marker on pVS2. Transformants were tested for increased phage resistance. In this way we identified three plasmids (pJW563, pJW565, and pJW566) which coded for R-M systems in W56 (23). These plasmids ranged in size from 11 to 25 kb. The efficiency of plating (EOP) for the isometric-headed phage p2 (16) varied from 10.sup.-2 to 10.sup.-3 for different plasmids. The existence of multiple R-M encoding plasmids in Lactococci's strains has previously been reported by Chopin's group in France (5, 13).
That multiple R-M-encoding plasmids can increase phage resistance was confirmed by stacking two-three plasmids (23, 24). The data in the following Table A show the efficiency of plating (EOP) of phage p2 on the various transformants, the numbers in parentheses in column 2 show the EOP of phage p2 with the R-M plasmid alone in L. lactis MG1614.
TABLE A Plasmid encoding R-M systems assembled in L. lactis and their effects on the EOP of phage p2 or jj50.sup.a. Transformants.sup.b Plasmid encoding R-M EOP MG1614 none 1 T128 pJW563 (10.sup.-3) + pJW565 (10.sup.-2) 10.sup.-5 T46 pJW563 (10.sup.-3) + pJW566 (2 .times. 10.sup.-2) 4 .times. 10.sup.-6 T45 pJW565 (10.sup.-2) + pJW566 (2 .times. 10.sup.-2) 10.sup.-3 T8 pJW563 (10.sup.-3) + pJW565 (10.sup.-2) + 3 .times. 10.sup.-6 pJW566 (2 .times. 10.sup.-2) J96 pJW563 (10.sup.-3) + pFV1001 (10.sup.-1) 10.sup.-5 J92 pJW563 (10.sup.-3) + pFV1201 (10.sup.-1) 10.sup.-5 J75 pJW563 (10.sup.-3) + pFV1001 (10.sup.-1) + 4 .times. 10.sup.-7 pTRK12 (10.sup.-1) .sup.a Phages p2 and jj50 were propagated on L. lactis MG1614[pVS2]. L. lactis MG1614 is a sm.sup.R, Opp.sup.d derivative of L. lactis MG1363 (46). .sup.b All transformants also harbored pVS2.
As shown in Table A, the effect of assembling R-M plasmids were additive in most cases. This supports the importance of R-M systems in the phage resistance of the TK5 starter. We did not obtain completely phage resistant strains, however, Sing and Klaenhammer (41) showed that in combination with other phage resistance mechanisms, e.g., abortive infection, R-M systems are powerful tools.
Transformant T1.1 (L. lacits MG1614+pJW563) and a transformant harbouring a plasmid pFW094 from L. lactis W9 (34) exhibited type II endonuclease activity showing that type II R-M systems can be plasmid encoded in Lactococcus lactis.
The ENases R.LlaAI and R.LlaBI from W9 and W56, respectively, were partially purified; and the recognition sequences for both ENases were identified by digesting well known DNA (pBR322/328, .lambda. DNA) with the respective ENases, treating the fragments with either the Klenow fragment of DNA polymerase or mung bean nuclease and ligating the resulting fragments into pBluescriptIISK+(Stratagene, La Jolla, Calif., USA) digested with EcoRV. By sequencing the junction fragments of the obtained clones, the recognition sequence of the respective ENases could be determined (34).
We found that R.LlaAI and R.LlaBI recognized 5'-.dwnarw.GATC-3' and 5'-C.dwnarw.TRYAG-3', respectively, digesting as indicated by the arrows. ENase R.LlaAI is therefore an isoschizomer of MboI from Moraxella bovis and DpnII from Streptococcus pneumoniae. R.LlaBI is an isoschizomer of SfcI from Enterococcus faecium. Identical ENase cleavage patterns were obtained in digests of pBluescriptIISK+, pBR322 and M13mp20 with R.LlaAI and MboI, and with R.LlaBI and SfcI, respectively.